Protein secretion pathways in the phylum Bacteroidetes

类杆菌属的成员是迄今为止人类肠道微生物群中最丰富的革兰氏阴性细菌。我们使用质谱仪检测了实验室条件下生长的类杆菌属中最常见的成员之一--脆弱杆菌的外膜蛋白质组和分泌组。虽然我们没有发现任何新的分泌途径，但我们发现这种生物使用的分泌策略与大肠杆菌等变形杆菌非常不同。脆弱芽孢杆菌缺乏在变形杆菌中广泛存在的许多途径(例如，II型、III型和IV型途径)，但同时产生多种I型分泌系统。I型途径的底物是未知的，并且不容易根据与变形杆菌中I型底物的蛋白质的同源性来预测。脆弱芽孢杆菌与变形杆菌也有很大的不同，因为它产生大量暴露在细胞表面的脂蛋白。我们目前正在使用各种方法来研究脂蛋白通过OM运输的机制(S)，并识别专门用于出口的特定脂蛋白的靶向信号。有趣的是，分泌组中发现的许多蛋白质与Genbank数据库中的蛋白质缺乏显著的同源性，推测它们具有新的功能。 在最近的一项研究中，我们研究了Fragiain的功能，这是一种暴露在脆弱芽孢杆菌细胞表面的、特征不佳的梭状芽胞杆菌疼痛样蛋白酶。令人惊讶的是，我们发现编码这种蛋白质的基因(Fpn)的破坏导致分泌体中&gt；100蛋白质水平的大幅下降，其中许多蛋白质被预测为脂蛋白。用纯化的Fragiain进行的实验提供了直接的证据，证明这种蛋白酶至少从细胞表面释放了这些蛋白质中的一部分。观察到，野生型细胞在共培养试验中击败了FPN-菌株，这也支持了强痛在细胞生理学中发挥重要作用的观点。最后，我们发现纯化的Fragiain改变了HT29肠上皮细胞的黏附特性。我们的结果表明，Fragiain是一种广谱的蛋白酶，不仅能催化蛋白质的广泛分泌，而且在定植过程中还有可能裂解宿主细胞的蛋白质。

Members of the genus Bacteroides are by far the most abundant Gram-negative bacteria in the human gut microbiome. We used mass spectrometry to examine the outer membrane (OM) proteome and secretome of one of the most commonly studied members of the Bacteroides genus, B. fragilis, grown under laboratory conditions. Although we did not identify any novel secretion pathways, we found that this organism uses a very different range of secretion strategies than Proteobacteria such as E. coli. B. fragilis lacks many of the pathways that are widespread among the Protebacteria (e.g., the type II, III and IV pathways) but produces multiple type I secretion systems simultaneously. The substrates of the type I pathways are unknown and cannot be easily predicted based on homology to proteins that are type I substrates in Proteobacteria. B. fragilis also differs dramatically from Proteobacteria in that it produces a large number of lipoproteins that are exposed on the cell surface. We are currently using a variety of methods to investigate the mechanism(s) by which lipoproteins are transported across the OM and to identify targeting signals that earmark specific lipoproteins for export. Interestingly, many of the proteins found in the secretome lack significant homology to proteins that are in the Genbank database and presumably have novel functions. In a recent study we examined the function of fragipain, a poorly characterized clostripain-like protease that is exposed on the cell surface of B. fragilis. Surprisingly, we found that disruption of the gene that encodes this protein (fpn) led to a strong reduction in the level of >100 proteins, many of which are predicted to be lipoproteins, in the secretome. Experiments performed with purified fragipain provided direct evidence that the protease releases at least some of these proteins from the cell surface. The observation that wild-type cells outcompeted an fpn- strain in co-cultivation assays also supported the notion that fragipain plays an important role in cell physiology. Finally, we found that purified fragipain altered the adhesive properties of HT29 intestinal epithelial cells. Our results suggest that fragipain is a broad-spectrum protease that not only catalyzes protein secretion on a wide scale but that also potentially cleaves host cell proteins during colonization.